The present invention is directed to the uses in separation of a class of membrane filters to which are attached ligands, substances capable of forming specific complexes with certain species present in a mixture. The specific agents or ligands are attached by chemical bonds to the inner pore surface of the membrane under pressure-driven conditions. The pore diameters of these membranes and their chemical nature are such as to allow for the coupling of a high concentration of these ligands on their inner surfaces, while still providing access to the solute molecules whose separation and purification is desired. These pores must be large enough to allow these soluble substances to be complexed without excessive steric hindrance. Further, the nature of these membranes is such that the excess and undesired components of the mixture can be readily washed out of the membrane under pressure-driven conditions, and then the complex separated and the desired substance displaced in a pure and concentrated state.
This invention teaches new and advantageous means of effecting separations for: analytical purposes; preparative purposes at the laboratory level; purposes of industrial production. It can be compared with the conventional processes of affinity chromatography which are described by several authors, particularly P. Cuatrecasas (J.Biol.Chem., 245, 3059 (1970)). This important technique is employed in biology and medicine and usually involves the use of insoluble gel-type beads of agarose, polyacrylamides or other polymers to which ligands capable of coupling to various molecules in a specific manner are attached. The attachment of ligands may be directly to the gel matrix, but it usually takes place through extended molecular or hydrocarbon chains which place the ligand at varying distances from the gel matrix backbone, the purpose of which is presumably to allow the ligand to come in close proximity to the active site of the molecule being separated. It has been postulated that the ligand must enter the "cleft" of a molecule such as an enzyme, and therefore the chain is a necessary requirement.
These fine beads are employed as specific adsorbents wherein complex mixtures are passed through a bed of such beads and complexes are formed between the ligand and certain molecular species present in the mixture. Then the other, non-coupled solutes present are displaced from the column with water or an appropriate solution in such way that the complex is undisturbed and in this manner a separation of the desired molecular species from the others is achieved. Following this washing procedure the complex is cleaved by passing through the column a soluble ligand as a displacing agent or by a solution of appropriate pH, salt concentration or solute composition, such as the use of urea, guanidine nitrate and the like. All of these techniques are well known in the technical literature.
This classical technique of affinity chromatography has proven to be very useful for the separation of very small amounts of specific substances present in complex mixtures. It has, however, suffered from a number of disadvantages. Among these is the slowness of the procedure and its extremely low capacity. For example, the amount of loading of the ligand on the beads is usually small and usually but a small fraction of the theoretical capacity of the ligand molecules is achieved. Further, since all of these processes are diffusion controlled and the rate of diffusion of proteins (the usual substances being separated) in the pores is very low, the entire procedure of loading can take several hours. The following procedure of washing is similarly quite slow because of the need for high molecular weight impurities to be desorbed and washed out of the bed of beads having fine pore diameters. Following this, the use of a displacement solution can frequently cause a deswelling of the beads and this further retards the rate of elution. Accordingly, the conventional procedures are characterized by a cycle time of many hours or days, and, where the amounts being isolated are of the order of milligrams, the slowness of the procedure results in highly labile molecules being partially or largely decomposed during the periods of the sorption, washing and elution steps.
Conventional affinity chromatography has been applied to the purification of a number of proteins, enzymes and other biologically important molecules. The ligands used include: specific competitive inhibitors; anti-enzymes; enzyme inhibitors. It has been believed by many investigators that it is important that the ligands be fixed at an appropriate and sufficient distance from the gel matrix backbone in order to be capable of binding in a specific manner. However, in more recent years it has been found that the nature of the role of the chain molecule may be more to enhance hydrophobic adsorptive processes in providing for the appropriate distance from the matrix. More recently it has been found that hydrocarbon chains of varying lengths alone can serve as ligands. They do not possess the high degree of selective affinity of enzyme inhibitors, as an example, but they do provide for the separation of related groups or classes of molecules of a biological nature, and these hydrophobic ligands have been employed also for purposes of affinity chromatography.
The teachings of the present invention are directed to novel means for preparing these affinity sorption systems, to the membranes used for these purposes and to a range of applications. These pressure-driven systems can be used for virtually all of the applications in which affinity chromatography gel systems have been used heretofore: they can also be used for high speed separations of an analytical nature, for preparation of materials which are unstable so their isolation must proceed rapidly, and for large-scale separations where a high capacity and high rate of turnover are important.